Evidence for a restoration of <scp>TLR2</scp> response in epidermal dendritic cells in atopic dermatitis by topical anti‐inflammatory therapy
Authors: Marie‐Sophie Philipp, Tim J. Nümm, Yuxuan Deng, Kazumasa Iwamoto, Nadine Herrmann, Thomas Bieber
Journal: Allergy (2023)

Patients suffering from atopic dermatitis (AD) show an attenuated immune response and imbalanced microbiome environment that favors Staphylococcus aureus (S. aureus).1 Bacterial products are recognized via Toll-like receptors (TLR) of the immune system leading to maturation of dendritic cells (DC).2 Recently, we showed that Langerhans cells (LC), DC in the epidermis, from ex vivo AD skin had an inhibited maturation response toward TLR2 stimulation when compared to healthy controls (HC).3 Topical anti-inflammatory treatment of AD reduces inflammation and the colonization with S. aureus.4 To test its impact on the TLR2 expression and function on epidermal DC, we analyzed DC freshly isolated from lesional skin of treated (topical steroids or tacrolimus for 2 days [median]) and untreated AD patients as well as HC (for methods see Appendix S1). LC of HC showed elevated TLR2 and similar CD83 and MHCII expression compared to AD, confirming our previous results (Figure 1). CD83 expression was lower in treated AD. CD80 expression was significantly higher in AD, as described,5 but independent of the treatment. TLR2 and maturation-associated marker expression on LC (Figure 1) and inflammatory dendritic epidermal cells (IDEC) (Figure S1) in AD skin failed to show significant differences upon treatment except for CD83 expression on LC. Upon anti-inflammatory treatment, only CD83 levels were significantly decreased. However, it has been reported that treatment for 1 week is able to reduce expression of maturation markers like CD80 and CD86 on LC.6 The short treatment duration in our study may explain differences in the expression. We further explored the ability of LC from treated AD patients to respond to TLR stimulus. Therefore, we cultured AD shave biopsy skin in the presence or absence of the TLR2-specific ligand Pam3Cys mimicking S. aureus-derived signals. LC from treated lesional AD skin showed a clear response to Pam3Cys with a downregulation of TLR2 and significant upregulation of CD83, while LC from untreated AD skin lack TLR2 response as described previously3 (Figure 2). A trend of MHCII and CD86 upregulation was observed in a limited number of experiments, though this has to be considered as preliminary. However, HC show an upregulation of MHCII under these conditions.3 Taken together we conclude that anti-inflammatory treatment restores the ability of epidermal LC to respond to TLR ligands and this is measurable on the molecular level shortly after treatment and before macroscopic clearance of the skin. We confirmed this by a separated analysis of patients treated for less than 4 days. (Figure S2). Additionally, IDEC from treated skin showed a similar response to TLR2 ligand Pam3Cys (Figures S3,S4), suggesting a general mechanism for the lack of TLR2 responsiveness and its restoration in epidermal DC in AD. In contrast to the tolerance observed in untreated AD skin, topical anti-inflammatory treatment seems to abrogate this tolerance leading to DC that are again capable of responding to stimulation. One explanation fitting the overall picture of AD is that LC are activated in situ due to the local microenvironment including impaired skin barrier, inflammation, and constant exposure to S. aureus, while treatment restores the functional capacity of DC. In addition, the disinfected skin still harbors dead S. aureus-derived particles that may influence the activation during the cell culture. However, one would expect that this is also the case for treated AD, but there was no sign of elevated activation in the control conditions. However, the skin's S. aureus load was not analyzed here. Another limit is the exclusion of migrated cells from analyses. Besides surface marker expression we analyzed the cytokine production. The production of IL-6 and IL-10 were enhanced in treated AD skin compared with untreated AD skin. By contrast, IL-18 production was reduced in treated patients (Figure S5). These levels in treated patients are similar to the production measured in healthy controls3 indicating that cytokine production is adjusted to a more healthy status after topical treatment. The mechanisms of the TLR restoration remain to be elucidated. In summary, no changes in the surface expression of TLR2 and maturation markers were observed in DC from lesional and briefly treated lesional AD except for the maturation marker CD83. Nevertheless, topical treatment seems to restore the TLR2 reactivity, where DC showed an increased expression of maturation markers and reduced TLR2 receptor expression upon TLR2 stimulation. Though it remains speculative if restauration of the TLR2-signaling is the initial starting point of skin improvement, our results strongly suggest that anti-inflammatory treatment is able to restore TLR2 signaling in DC in the lesion rapidly and before phenotypical changes are detected. Marie-Sophie Philipp performing experiments, data analyses and interpretation, statistical analyses, and writing the manuscript. Tim Nümm performing experiments, data analyses, interpretation, and initial manuscript draft. Kazumasa Iwamoto performing experiments and data analyses. Yuxuan Deng data interpretation, critical reviewing of the manuscript, and performing experiments. Nadine Herrmann supervision, conceptional input, critical reviewing of the manuscript, and revision of the manuscript. Thomas Bieber conceptional design, supervision, patients recruitment, and critical reviewing of the manuscript. We thank all patients for their skin donation, Dr. Lisa Braun, Dr. Sophie Kläschen, and Dr. Laura Maintz of the Department of Dermatology and Allergy, University of Bonn, Germany, for performing the shave biopsy and Helene Kirins and Sylvia Schnautz for the expert technical assistance. We thank Dr. K. Walgenbach of the Department of Plastic and Aesthetic Surgery, University of Bonn, Germany, for providing us with normal skin samples. Open Access funding enabled and organized by Projekt DEAL. This study was supported by the Christine Kühne—Center for Allergy Research and Education (CK-CARE). Tim J. Nümm, Marie-Sophie Philipp, Yuxuan Deng, and Nadine Herrmann were supported by CK-CARE. Thomas Bieberer was speaker, and/or consultant, and/or investigator for: AbbVie, Allmiral, AnaptysBio, Arena, Asana Biosciences, Astellas, BioVerSys, Böhringer Ingelheim, Celgene, Daiichi Sankyo, Dermavant/Roivant, DermTreat, DS Pharma, RAPT/ FLX Bio, Galapagos/MorphoSys, Galderma, Glenmark, GSK, Incytes, Kymab, LEO, Lilly, L'Oréal, MenloTx, Novartis, Pfizer, Pierre Fabre, Sanofi/Regeneron, and UCB. Thomas Bieber is founder of the nonprofit biotech company “Davos Biosciences”. All other authors have no conflicts of interest to declare. Research data are not shared. Figure S1 Figure S2 Figure S3 Figure S4 Figure S5 Appendix S1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.